• Korean Journal of Fisheries and Aquatic Sciences
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• v.24 no.5
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• pp.273-288
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• 1991

To elucidate the physiological and biochemical differences between chondrichthyes and osteichthyes, the properties of the specific digestive enzymes in cat-shark, Cephaloscyllium umbratile, and mackerel, Scomber japonicus, were studied. Homogenous trypsin proved through the disc-electrophoresis, SDS-PAG electrophoresis and gel filtration was obtained from the pancreas of cat-shark by $50-70\%$ saturated ammonium sulphate fractionation, DEAE-Sephadex A-50 column chromatography, benzamidine-Sepharose 6B affinity chromatography and Sephadex G-75-120 gel filtration. Two types of trypsins were also obtained from the pyloric caeca of mackerel by $30-70\%$ saturated ammonium sulphate fractionation and the slightly modified procedure from the method adopted in the purification of cat-shark trypsin. The two trypsins, designated trypsin A and B, were proved their homogeneity by disc- and SDS-PAG electrophoresis and gel filtration. The molecular weights of the trypsins were estimated to be 31,700 for cat-shark trypsin, 30,000 for mackerel trypsin A and 29,000 for mackerel trypsin B by SDS-PAG electrophoresis, but those were estimated to be 21,500 for cat-shark trypsin, 23,700 for mackerel trypsin A and 21,500 for mackerel trypsin B by gel filtration. The trypsins exhibited their optimum conditions at pH 9.0 and on temperature ranged from $45^{\circ}C\;to\;50^{\circ}C$ for cat-shark, and at pH 8.0 and a temperature of $50^{\circ}C$ for mackerel trypsin A and B, respectively. The cat-shark trypsin was stable at pH 10.0 and the temperature below $10^{\circ}C$, whereas the mackerel trypsin A and B, were stable in the range over pH 7.0 to pH 9.0 below $10^{\circ}C$ and at pH 8.0 below $35^{\circ}C$, respectively. The mackerel trypsins were severely inhibited by some heavy metal ions such as $Ag^{2+},\;Cu^{2+}\;and\;Hg^{2+}$ compared to cat-shark trypsin. All of the enzymes were also inhibited by antipain, leupeptin, TLCK(tosyllysine chloromethyl ketone) and SBTI(soybean trypsin inhibitor) remarkably. The inhibitory effects of PMSF(phenylmethane sulphonylfluoride), DFP(diisopropyl fluorophosphate) and benzamidine were indicated that these enzymes belong to serine-proteases.

• Clean Technology
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• v.13 no.1 s.36
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• pp.54-63
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• 2007

Even traces of CO in the hydrogen-rich feed gas to proton exchange membrane fuel cells (PEMFC) poison the platinum anode electrode and dramatically decrease the power output. In this work, a variety of catalytic materials consisting of $Cu/Ce_xZr_{1-x}O_2$, (x = 0.0-1.0) were synthesised, characterized and tested for CO oxidation and preferential oxidation of CO (PROX). These catalysts prepared by hydrothermal and deposition-precipitation methods. The catalysts were characterized by XRD, XRF, SEM, BET, $N_2O$ titration and oxygen storage capacity (OSC) measurement. The effects of composition of the support and degree of excess oxygen were investigated fur activity and $CO_2$ selectivity with different temperatures. The composition of the support markedly influenced the PROX activity. Among the various $Cu/Ce_xZr_{1-x}O_2$ catalysts having different composition, $Cu/Ce_{0.9}Zr_{0.1}O_2$ and $Cu/Ce_{0.7}Zr_{0.3}O_2$ showed the highest activities (>99%) and selectivities (ca.50%) in the temperature range of $150{\sim}160^{\circ}C$. It was found that by using of $Ce_xZr_{1-x}O_2$ mixed oxide support which possesses a high oxygen storage capacity, oxidation-reduction activity of Cu-based catalyst was improved, which resulted in the increase of catalytic activity and selectivity of CO oxidation in excess $H_2$ environments.

• Journal of Life Science
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• v.25 no.3
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• pp.307-316
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• 2015

Beetle larvae have been used as a traditional medicine to treat various human liver diseases. To prove the liver protective function of Allomyrina dichotoma larvae (ADL), we induced liver damage by the intraperitoneal injection of a hepatotoxic reagent, diethylnitrosamine (DEN), to C3H/HeN male mice and orally administered freeze-dried ADL powder. ADL powder lessened DEN-induced hepatotoxicity considering the reduced signs of acute and chronic hepatotoxicities, such as the ALP level in the blood serum, TUNEL-positive hepatocytes, ductural reactions, steatotic hepatocytes, and collagen deposition of the Masson’s trichrome staining. In addition to hepatoprotection, the anti-cancer activity of ADL has been examined. The ADL powder was extracted with ethanol and then fractionated with hexane, ethyl acetate, and water by a solvent partition technique. The ethyl acetate fraction showed cytotoxicity to various cancer cells through induction of apoptosis and necrosis, as well as the perturbed metabolism of the cancer cell to trigger autophagy. Collectively, ADL contains bioactive substances that can protect hepatocytes from toxic chemicals and trigger cell death in cancer cells. Thus, further purification and analyses of ADL fractions could lead to the identification of novel bioactive compounds.

• Proceedings of the Korean Society for Food Science of Animal Resources Conference
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• 2004.05a
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• pp.89-119
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• 2004

This study was conducted to isolate lactobacilli having probiotic characteristics to be used as health adjuncts with fermented milk products. Acid tolerant strains were selected in Lactobacilli MRS broth adjusted to pH 4.0 from 80 healthy persons (infants, children and adults). And bile tolerant strains were examined in Lactobacilli MRS broth in which 1.0% bile salt was added. By estimation above characteristics, the strains No. 27, which was isolated from adult feces, was selected and identified as Lactobacillus salivarius subsp. salivarius based on carbohydrate fermentation and 16S rDNA sequencing. It was used as a probiotic strain in fermented milk products. The pH of fermented milk decreased from pH 6.7 to 5.0 and titratable acidity increased from 0.3% to 1.0% by L. salivarius subsp. salivarius (isolation strain 20, 35, and 37), when incubated for 36 h at $37^{\circ}C$. The number of viable cell counts of fermented milk was maximized at this incubation condition. The SDS-PAGE evidenced no significant change of casein but distinct changes of whey protein were observed by isolated L. salivarius subsp. salivarius for titratable acidity being incubated by $0.9{\sim}1.0%$ at $37^{\circ}C$. All of the strains produced 83.43 to 131.96 mM of lactic acid and 5.39 to 26.85 mM of isobutyric acid in fermented products. The in vitro culture experiment was performed to evaluate ability to reduce cholesterol levels and antimicrobial activity in the growth medium. The selected L. salivarius subsp. salivarius reduced $23{\sim}38%$ of cholesterol content in lactobacilli MRS broth during bacterial growth for 24 hours at $37^{\circ}C$. All of the isolated L. salivarius subsp. salivarius had an excellent antibacterial activity with $15{\sim}25$ mm of inhibition zone to E. coli KCTC1039, S. enteritidis KCCM3313, S. typhimurium M-15, and S. typhimurium KCCM40253 when its pH had not been adjusted. Also, all of the isolated L. salivarius subsp. salivarius had partial inhibition zone to E. coli KCTC1039, E. coli KCTC0115 and S. enteritidis KCCM3313 when it had been adjusted to pH 5.7. The selected strains were determined to have resistances of twelve antibiotic. Strains 27 and 35 among the L. salivarius subsp. salivarius showed the highest resistance to the antibiotics. Purified ${\alpha}$-galactosidase was obtained by DEAE-Sephadex A-50 ion exchange chromatography, Mono-Q ion exchange chromatography and HPLC column chromatography from L. salivarius subsp. salivarius 27. The specific activity of the purified enzyme was 8,994 units/mg protein, representing an 17.09 folds purification of the original cell crude extract. The molecular weight of enzyme was identified about 53,000 dalton by 12% SDS-PAGE. Optimal temperature and pH for activity of this enzyme were $40^{\circ}C$ and 7.0 respectively. The enzyme was found to be stable between 25 and $50^{\circ}C$. ${\alpha}$-galactosidase activity was lost rapidly below pH 5.0 and above pH 9.0. This enzyme was liberated galactose from melibiose, raffinose, and stachyose, and also the hydrolysis rate of substrate was compound by HPLC. These results indicated that some of the L. salivarius subsp. salivarius (strain 27 and 35) are considered as effective probiotic strains with a potential for industrial applications, but the further study is needed to establish their use as probiotics in vivo.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.27 no.5
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• pp.482-494
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• 1994

Most of carotenoprotein complexes have been extracted by using buffered solutions. However, in this study carotenoprotein from the muscle of Blue mussel(Mytilus edulis) was extracted by a detergent such as Triton X-100. It was purified and characterized by $20\%$ (w/v) $(NH_4)_2SO_4$, DEAE-cellulose ion exchange and Sephacryl S-300 gel filtration. The carotenoprotein(${\lambda}_{max}=462nm$) had an approximate M. W. of 372KDa(gel filtration). SDS-PAGE analysis of the carotenoprotein indicated the presence of four polypeptides of 60KDa($23.70\%$), 46.9KDa($9.14\%$), 26KDa($49.14\%$) and 13KDa($18.02\%$). Carotenoprotein denaturated by treatment with SDS to a final concentration of $0.2\%$ (w/v) caused a hypsochromic shift of ${\lambda}_{max}$ from 462nm to 456nm. The carotenoprotein contained lipids as structure units. The amino acid composition of the carotenoprotein contained large essential amino acid amounts of $62.8\%$, and the content of threonine($35.9\%$) was higher than other amino acids, but histidine, methionine and proline were not present. In the carotenoprotein, the major fatty acids were $C_{16:4},\;C_{16:0},\;C_{20:5}\;and\;C_{22:6}$. The percentages of polyunsaturated fatty acids($62.4\%$) were higher compared to other fatty acids(saturated fatty acids $19.6\%$, monounsaturated fatty acids $18.0\%$). Carotenoid was extracted from the carotenoprotein by acetone and it was separated into five different components by preparative TLC(benzene:petroleum ether:acetone=69:17:14). The major components of carotenoid were mytiloxanthin($74.79\%$) and 3,4,3'- trihydroxy-7',8'-didehydro-${\beta}$-carotene($18.26\%$), and they were at least presented as prosthetic groups of carotenoprotein.

• Tuberculosis and Respiratory Diseases
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• v.45 no.1
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• pp.36-44
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• 1998

Background: IFN-$\gamma$ is known to activate mononuclear phagocytes and to mediate host defense mechanism against some intracellular microorganisms, but little is known about anti-mycobacterial activity and mechanism of IFN-$\gamma$ in human. In this study, we investigated the role of IFN-$\gamma$ in the pathogenesis of tuberculosis by observing the effect of IFN-$\gamma$ on the phagocytosis of M.tuberculosis(MTB) and on the production of TNF-$\alpha$ by human pulmonary alveolar macrophage. Method: Pulmonary alveolar macrophage(PAM) were prepared with adhesion purification method from bronchoalveolar lavage fluid obtained from 8 persorn without active lung lesion and cultured($1{\times}10^6cells/ml$) with MTB($3{\times}10^7$ bacteria/ml) with or without IFN-$\gamma$(300U/ml), LPS(0.5ug/ml) and autologous serum(10%). After 2 hours, the percentage of PAM-phagocytosed MTB was counted after AFB staining(modified Kynion method). TNF-$\alpha$ production by PAM stimulated by IFN-$\gamma$(300U/ml), MTB($1{\times}10^6bacteria/ml$) and LPS(0.5ug/ml) for 24hours was measured in culture supernatant using ELISA method. The degree of phagocytosis of MTB by PAM stimulated with IFN-$\gamma$(300U/ml) and LPS(0.5ug/ml) for 24hours was also investigated. Results: IFN-$\gamma$ did not influence the phagocytosis of MTB by PAM(percentage of PAM-phagocytosed MTB: control: $22.1{\pm}4.9$, IFN-$\gamma$: $20.3{\pm}5.3$) and did not increase TNF-$\alpha$ production by PAM (control: $21{\pm}38pg/ml$, IFN-$\gamma$: $87{\pm}106pg/ml$), and the degree of phagocytosis of MTB by PAM pre-stimulated with IFN-$\gamma$ for 24 hours, was not increased (control: $24.5{\pm}9.5$, IFN-$\gamma$: $23.4{\pm}10.1$). Conclusion: IFN-$\gamma$ does not influence on the phagocytosis of MTB and TNF-$\alpha$ production by PAM.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.28 no.5
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• pp.375-395
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• 2006

Purpose: Lingual nerve (LN) damage may be caused by either tumor resection or injury such as wisdom tooth extraction, Although autologous nerve graft is sometimes used to repair the damaged nerve, it has the disadvantage of necessity of another operation for nerve harvesting. Moreover, the results of nerve grafting is not satisfactory. The nerve growth factor (NGF) is well-known to play a critical role in peripheral nerve regeneration and its local delivery to the injured nerve has been continuously tried to enhance nerve regeneration. However, its application has limitations like repeated administration due to short half life of 30 minutes and an in vivo delivery model must allow for direct and local delivery. The aim of this study was to construct a well-functioning $rhNGF-{\beta}$ adenovirus for the ultimate development of improved method to promote peripheral nerve regeneration with enhanced and extended secretion of hNGF from the injured nerve by injecting $rhNGF-{\beta}$ gene directly into crush-injured LN in rat model. Materials and Methods: $hNGF-{\beta}$ gene was prepared from fetal brain cDNA library and cloned into E1/E3 deleted adenoviral vector which contains green fluorescence protein (GFP) gene as a reporter. After large scale production and purification of $rhNGF-{\beta}$ adenovirus, transfection efficiency and its expression at various cells (primary cultured Schwann cells, HEK293 cells, Schwann cell lines, NIH3T3 and CRH cells) were evaluated by fluorescent microscopy, RT-PCR, ELISA, immunocytochemistry. Furthermore, the function of rhNGF-beta, which was secreted from various cells infected with $rhNGF-{\beta}$ adenovirus, was evaluated using neuritogenesis of PC-12 cells. For in vivo evaluation of efficacy of $rhNGF-{\beta}$ adenovirus, the LNs of 8-week old rats were exposed and crush-injured with a small hemostat for 10 seconds. After the injury, $rhNGF-{\beta}$ adenovirus($2{\mu}l,\;1.5{\times}10^{11}pfu$) or saline was administered into the crushed site in the experimental (n=24) and the control group (n=24), respectively. Sham operation of another group of rats (n=9) was performed without administration of either saline or adenovirus. The taste recovery and the change of fungiform papilla were studied at 1, 2, 3 and 4 weeks. Each of the 6 animals was tested with different solutions (0.1M NaCl, 0.1M sucrose, 0.01M QHCl, or 0.01M HCl) by two-bottle test paradigm and the number of papilla was counted using SEM picture of tongue dorsum. LN was explored at the same interval as taste study and evaluated electro-physiologically (peak voltage and nerve conduction velocity) and histomorphometrically (axon count, myelin thickness). Results: The recombinant adenovirus vector carrying $rhNGF-{\beta}$ was constructed and confirmed by restriction endonuclease analysis and DNA sequence analysis. GFP expression was observed in 90% of $rhNGF-{\beta}$ adenovirus infected cells compared with uninfected cells. Total mRNA isolated from $rhNGF-{\beta}$ adenovirus infected cells showed strong RT-PCR band, however uninfected or LacZ recombinant adenovirus infected cells did not. NGF quantification by ELISA showed a maximal release of $18865.4{\pm}310.9pg/ml$ NGF at the 4th day and stably continued till 14 days by $rhNGF-{\beta}$ adenovirus infected Schwann cells. PC-12 cells exposed to media with $rhNGF-{\beta}$ adenovirus infected Schwann cell revealed at the same level of neurite-extension as the commercial NGF did. $rhNGF-{\beta}$ adenovirus injected experimental groups in comparison to the control group exhibited different taste preference ratio. Salty, sweet and sour taste preference ratio were significantly different after 2 weeks from the beginning of the experiment, which were similar to the sham group, but not to the control group.

• Tuberculosis and Respiratory Diseases
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• v.44 no.3
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• pp.601-610
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• 1997

Background : Monocytes/macrophages play a central role in determining the host response during Gram-negative infection through secretion of a variety of mediators after stimulation of LPS. Even though cytokine production has been shown to play an important role in host defense during sepsis, cytokine release may also lead to tissue injury. Thus, regulation of macrophage response to LPS is critical for host survival during Gram-negative sepsis. In animals exposed to nonlethal doses of endotoxin, a characteristic hyporesponsiveness to subsequent administration of endotoxin has been observed. This phenomenon was known as 'LPS tolerance'. However, little information is available regarding the underlying mechanism of LPS tolerance. Method : Peripheral blood monocyte(PBMC) was isolated from peripheral blood of normal volunteers by adhesion purification method. To evaluate the conditions to obtain LPS tolerance, preculture was carried out with LPS at 10ng/ml for 24 hours. For stimulation, culture plates were washed two times and were stimulated with LPS at $1{\mu}g/ml$ for 4, 6 and 26 hours. To assess the underlying mechanisms of LPS tolerance, autologous serum, PMA, anti-CD14 Ab, Indomethacin or $PGF_2$ were added to preculture solution respectively. Cytokine concentrations in culture supernatants were measured using ELISA for TNF-$\alpha$ and IL-8 and mRNA of TNF-$\alpha$ and IL-8 were determined by Northern blot analysis. Results : The exposure of PBMC to low dose of LPS suppressed the cytokine production and mRNA expression of TNF-$\alpha$, but not IL-8. Anti-CD14 Ab partially recovered production of TNF-$\alpha$ which was suppressed by preculture with low dose LPS. The preculture with PMA induces LPS tolerance, as preculture with low dose LPS. Conclusion : LPS tolerance to TNF-$\alpha$ is regulated pretranslationally and is influenced by protein kinase C pathway and CD14.

• Korean Journal of Food Science and Technology
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• v.7 no.4
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• pp.229-237
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• 1975

An attempt was made to utilize the enzyme produced by Asp. oryzae as meat tenderizer. The production, purification, and various properties of proteinase produced by Asp. oryzae were investigated. Results obtained are as follow; 1. A strain which had the highest proteolytic activity was selected among 9 Aspergillus species. 2. Culture medium consisted of wheat bran 10g, 2% glucose, 0.03% urea and 0.1% $MgSO_4$ (pH 6.5). Mold was incubated at $30^{\circ}C$ for 3 days. 3. Enzyme extract from culture medium were fractionated with ammonium sulfate and purified by Sephadex G-75 column chromatography. 4. When pH of reaction mixture was controlled, maximal activity of proteinase by Asp. oryzae was obtained at pH 3, pH 6.6, $8.4{\sim}8.5$ and pH 10.0 to 10.5. Those results were interpreted to show that enzyme consists of acid proteinase, neutral proteinase and alkaline proteinase. Enzyme was stable at pH 6 to 10. 5. Opt. temperature for proteinase activity was $50^{\circ}C$, but enzyme was stable up to $40^{\circ}C$. 6. The proteinase was inhibited by $Ag^+$. It was also inhibited by EDTA. 7. When myofibrillar proteins were treated by proteinase from Asp. oryzae, ATPase activities of myofibrillar proteins changed remarkably. Accordingly, it was concluded that proteinase produced by Asp. oryzae were able to be used as meat tenderizer.

• Journal of Life Science
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• v.20 no.7
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• pp.1107-1112
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• 2010

In this study, the stable maintenance of bioactivity in alkaline ionized water (AIW) and antibacterial effects of AIW were evaluated to confirm benefits of AIW. As controls, purified water (PW) and tap drinking water (DW) were used. The pH and ORP (oxidation-reduction potential) of AIW, PW and DW used were 9.5 and 120 mV, 7.2 and 144 mV, and 7.3 and 564 mV, respectively. High level of minerals was observed in DW (DW>AIW>PW of mineral contents). Concentrations of $Ca^{++}$ and $Na^+$ in DW were 14.5, and 8.4 mg/l, respectively, while no $Ca^{++}$, $Mg^{++}$, $K^+$, and $Na^+$ were detected in PW. Evaluation of antioxidant activities for AIW, PW and DW showed that the waters did not act as antioxidants. However, the DPPH (1,1-diphenyl-2-picryl hydrazyl) or superoxide radical scavenging activities or reducing power of vitamin C were stably maintained in AIW and PW, though not in DW, against heat treatment ($60^{\circ}C$) or vigorous shaking (120 rpm) at $37^{\circ}C$. Similarly, after aspirin treatment at $60^{\circ}C$ for 1 hr, the antithrombosis activity in PW and AIW was 62.6% and 55.3%, while that of DW was 52.1%. Furthermore, cell growth analysis and viable cell count of Escherichia coli H7:O157 in PW, AIW and DW showed that AIW and DW, not DW, have antibacterial activities. Our results suggest that the state of water, for example pH, ORP and mineral contents of water, should be considered in medicine or food industries, and that AIW has high potential for utilization in various fields.